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Phospholipase Cgamma/diacylglycerol-dependent activation of beta2-chimaerin restricts EGF-induced Rac signaling.

ABSTRACT: Although receptor-mediated regulation of small G-proteins and the cytoskeleton is intensively studied, the mechanisms for attenuation of these signals are poorly understood. In this study, we have identified the Rac-GAP beta2-chimaerin as an effector of the epidermal growth factor receptor (EGFR) via coupling to phospholipase Cgamma (PLCgamma) and generation of the lipid second messenger diacylglycerol (DAG). EGF redistributes beta2-chimaerin to promote its association with the small GTPase Rac1 at the plasma membrane, as determined by FRET. This relocalization and association with Rac1 were impaired by disruption of the beta2-chimaerin C1 domain as well as by PLCgamma1 RNAi, thus defining beta2-chimaerin as a novel DAG effector. On the other hand, GAP-deficient beta2-chimaerin mutants show enhanced translocation and sustained Rac1 association in the FRET assays. Remarkably, RNAi depletion of beta2-chimaerin significantly extended the duration of Rac activation by EGF, suggesting that beta2-chimaerin serves as a mechanism that self-limits Rac activity in response to EGFR activation. Our results represent the first direct evidence of divergence in DAG signaling downstream of a tyrosine-kinase receptor via a PKC-independent mechanism.

SUBMITTER: Wang H

PROVIDER: S-EPMC1462969 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A novel cross-talk in diacylglycerol signaling: the Rac-GAP beta2-chimaerin is negatively regulated by protein kinase Cdelta-mediated phosphorylation.

Project description:Although the family of chimaerin Rac-GAPs has recently gained significant attention for their involvement in development, cancer, and neuritogenesis, little is known about their molecular regulation. Chimaerins are activated by the lipid second messenger diacylglycerol via their C1 domain upon activation of tyrosine kinase receptors, thereby restricting the magnitude of Rac signaling in a receptor-regulated manner. Here we identified a novel regulatory mechanism for beta2-chimaerin via phosphorylation. Epidermal growth factor or the phorbol ester phorbol 12-myristate 13-acetate caused rapid phosphorylation of beta2-chimaerin on Ser(169) located in the SH2-C1 domain linker region via protein kinase Cdelta, which retained beta2-chimaerin in the cytosol and prevented its C1 domain-mediated translocation to membranes. Furthermore, despite the fact that Ser(169) phosphorylation did not alter intrinsic Rac-GAP activity in vitro, a non-phosphorylatable beta2-chimaerin mutant was highly sensitive to translocation, and displayed enhanced association with activated Rac, enhanced Rac-GAP activity, and anti-migratory properties when expressed in cells. Our results not only revealed a novel regulatory mechanism that facilitates Rac activation, but also identified a novel mechanism of cross-talk between diacylglycerol receptors that restricts beta2-chimaerin relocalization and activation.

| S-EPMC2878072 | biostudies-literature

Coordinated activation of the Rac-GAP β2-chimaerin by an atypical proline-rich domain and diacylglycerol.

Project description:Chimaerins, a family of GTPase activating proteins for the small G-protein Rac, have been implicated in development, neuritogenesis and cancer. These Rac-GTPase activating proteins are regulated by the lipid second messenger diacylglycerol generated by tyrosine kinases such as the epidermal growth factor receptor. Here we identify an atypical proline-rich motif in chimaerins that binds to the adaptor protein Nck1. Unlike most Nck1 partners, chimaerins bind to the third SH3 domain of Nck1. This association is mediated by electrostatic interactions of basic residues within the Pro-rich motif with acidic clusters in the SH3 domain. Epidermal growth factor promotes the binding of β2-chimaerin to Nck1 in the cell periphery in a diacylglycerol-dependent manner. Moreover, β2-chimaerin translocation to the plasma membrane and its peripheral association with Rac1 requires Nck1. Our studies underscore a coordinated mechanism for β2-chimaerin activation that involves lipid interactions via the C1 domain and protein-protein interactions via the N-terminal proline-rich region.

| S-EPMC3700536 | biostudies-literature

T cell receptor-dependent tyrosine phosphorylation of beta2-chimaerin modulates its Rac-GAP function in T cells.

Project description:The actin cytoskeleton has an important role in the organization and function of the immune synapse during antigen recognition. Dynamic rearrangement of the actin cytoskeleton in response to T cell receptor (TCR) triggering requires the coordinated activation of Rho family GTPases that cycle between active and inactive conformations. This is controlled by GTPase-activating proteins (GAP), which regulate inactivation of Rho GTPases, and guanine exchange factors, which mediate their activation. Whereas much attention has centered on guanine exchange factors for Rho GTPases in T cell activation, the identity and functional roles of the GAP in this process are largely unknown. We previously reported beta2-chimaerin as a diacylglycerol-regulated Rac-GAP that is expressed in T cells. We now demonstrate Lck-dependent phosphorylation of beta2-chimaerin in response to TCR triggering. We identify Tyr-153 as the Lck-dependent phosphorylation residue and show that its phosphorylation negatively regulates membrane stabilization of beta2-chimaerin, decreasing its GAP activity to Rac. This study establishes the existence of TCR-dependent regulation of beta2-chimaerin and identifies a novel mechanism for its inactivation.

| S-EPMC2670141 | biostudies-literature

p23/Tmp21 differentially targets the Rac-GAP beta2-chimaerin and protein kinase C via their C1 domains.

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| S-EPMC2854097 | biostudies-literature

?3-chimaerin, a novel member of the chimaerin Rac-GAP family.

Project description:Chimaerins are a family of diacylglycerol- and phorbol ester-regulated GTPase activating proteins (GAPs) for the small G-protein Rac. Extensive evidence indicates that these proteins play important roles in development, axon guidance, metabolism, cell motility, and T cell activation. Four isoforms have been reported to-date, which are products of CHN1 (?1- and ?2-chimaerins) and CHN2 (?1- and ?2-chimaerins) genes. Although these gene products are assumed to be generated by alternative splicing, bioinformatics analysis of the CHN2 gene revealed that ?1- and ?2-chimaerins are the products of alternative transcription start sites (TSSs) in different promoter regions. Furthermore, we found an additional TSS in CHN2 gene that leads to a novel product, which we named ?3-chimaerin. Expression profile analysis revealed predominantly low levels for the ?3-chimaerin transcript, with higher expression levels in epididymis, plasma blood leucocytes, spleen, thymus, as well as various areas of the brain. In addition to the prototypical SH2, C1, and Rac-GAP domains, ?3-chimaerin has a unique N-terminal domain. Studies in cells established that ?3-chimaerin has Rac-GAP activity and is responsive to phorbol esters. The enhanced responsiveness of ?3-chimaerin for phorbol ester-induced translocation relative to ?2-chimaerin suggests differential ligand accessibility to the C1 domain.

| S-EPMC3969763 | biostudies-literature

Characterization of the Rac-GAP (Rac-GTPase-activating protein) activity of beta2-chimaerin, a 'non-protein kinase C' phorbol ester receptor.

Project description:The regulation and function of beta2-chimaerin, a novel receptor for the phorbol ester tumour promoters and the second messenger DAG (diacylglycerol), is largely unknown. As with PKC (protein kinase C) isoenzymes, phorbol esters bind to beta2-chimaerin with high affinity and promote its subcellular distribution. beta2-Chimaerin has GAP (GTPase-activating protein) activity for the small GTP-binding protein Rac1, but for not Cdc42 or RhoA. We show that acidic phospholipids enhanced its catalytic activity markedly in vitro, but the phorbol ester PMA had no effect. beta2-Chimaerin and other chimaerin isoforms decreased cellular levels of Rac-GTP markedly in COS-1 cells and impaired GTP loading on to Rac upon EGF (epidermal growth factor) receptor stimulation. Deletional and mutagenesis analysis determined that the beta2-chimaerin GAP domain is essential for this effect. Interestingly, PMA has a dual effect on Rac-GTP levels in COS-1 cells. PMA increased Rac-GTP levels in the absence of a PKC inhibitor, whereas under conditions in which PKC activity is inhibited, PMA markedly decreased Rac-GTP levels and potentiated the effect of beta2-chimaerin. Chimaerin isoforms co-localize at the plasma membrane with active Rac, and these results were substantiated by co-immunoprecipitation assays. In summary, the novel phorbol ester receptor beta2-chimaerin regulates the activity of the Rac GTPase through its GAP domain, leading to Rac inactivation. These results strongly emphasize the high complexity of DAG signalling due to the activation of PKC-independent pathways, and cast doubts regarding the selectivity of phorbol esters and DAG analogues as selective PKC activators.

| S-EPMC1223697 | biostudies-other

Disruption of TrkB-mediated phospholipase Cgamma signaling inhibits limbic epileptogenesis.

Project description:The BDNF receptor, TrkB, is critical to limbic epileptogenesis, but the responsible downstream signaling pathways are unknown. We hypothesized that TrkB-dependent activation of phospholipase Cgamma1 (PLCgamma1) signaling is the key pathway and tested this in trkB(PLC/PLC) mice carrying a mutation (Y816F) that uncouples TrkB from PLCgamma1. Biochemical measures revealed activation of both TrkB and PLCgamma1 in hippocampi in the pilocarpine and kindling models in wild-type mice. PLCgamma1 activation was decreased in hippocampi isolated from trkB(PLC/PLC) compared with control mice. Epileptogenesis assessed by development of kindling was inhibited in trkB(PLC/PLC) compared with control mice. Long-term potentiation of the mossy fiber-CA3 pyramid synapse was impaired in slices of trkB(PLC/PLC) mice. We conclude that TrkB-dependent activation of PLCgamma1 signaling is an important molecular mechanism of limbic epileptogenesis. Elucidating signaling pathways activated by a cell membrane receptor in animal models of CNS disorders promises to reveal novel targets for specific and effective therapeutic intervention.

| S-EPMC2872928 | biostudies-literature

Membrane environment exerts an important influence on rac-mediated activation of phospholipase Cγ2.

Project description:We performed analyses of the molecular mechanisms involved in the regulation of phospholipase Cγ2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLCγ2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLCγ2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation.

| S-EPMC3067904 | biostudies-literature

Myeloproliferative disorder FOP-FGFR1 fusion kinase recruits phosphoinositide-3 kinase and phospholipase Cgamma at the centrosome.

Project description:BackgroundThe t(6;8) translocation found in rare and agressive myeloproliferative disorders results in a chimeric gene encoding the FOP-FGFR1 fusion protein. This protein comprises the N-terminal region of the centrosomal protein FOP and the tyrosine kinase of the FGFR1 receptor. FOP-FGFR1 is localized at the centrosome where it exerts a constitutive kinase activity.ResultsWe show that FOP-FGFR1 interacts with the large centrosomal protein CAP350 and that CAP350 is necessary for FOP-FGFR1 localisation at centrosome. FOP-FGFR1 activates the phosphoinositide-3 kinase (PI3K) pathway. We show that p85 interacts with tyrosine 475 of FOP-FGFR1, which is located in a YXXM consensus binding sequence for an SH2 domain of p85. This interaction is in part responsible for PI3K activation. Ba/F3 cells that express FOP-FGFR1 mutated at tyrosine 475 have reduced proliferative ability. Treatment with PI3K pathway inhibitors induces death of FOP-FGFR1 expressing cells. FOP-FGFR1 also recruits phospholipase Cgamma1 (PLCgamma1) at the centrosome. We show that this enzyme is recruited by FOP-FGFR1 at the centrosome during interphase.ConclusionThese results delineate a particular type of oncogenic mechanism by which an ectopic kinase recruits its substrates at the centrosome whence unappropriate signaling induces continuous cell growth and MPD.

| S-EPMC2373309 | biostudies-literature

The distinct roles of Ras and Rac in PI 3-kinase-dependent protrusion during EGF-stimulated cell migration.

Project description:Cell migration involves the localized extension of actin-rich protrusions, a process that requires Class I phosphoinositide 3-kinases (PI 3-kinases). Both Rac and Ras have been shown to regulate actin polymerization and activate PI 3-kinase. However, the coordination of Rac, Ras and PI 3-kinase activation during epidermal growth factor (EGF)-stimulated protrusion has not been analyzed. We examined PI 3-kinase-dependent protrusion in MTLn3 rat adenocarcinoma cells. EGF-stimulated phosphatidyl-inositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] levels showed a rapid and persistent response, as PI 3-kinase activity remained elevated up to 3 minutes. The activation kinetics of Ras, but not Rac, coincided with those of leading-edge PtdIns(3,4,5)P(3) production. Small interfering RNA (siRNA) knockdown of K-Ras but not Rac1 abolished PtdIns(3,4,5)P(3) production at the leading edge and inhibited EGF-stimulated protrusion. However, Rac1 knockdown did inhibit cell migration, because of the inhibition of focal adhesion formation in Rac1 siRNA-treated cells. Our data show that in EGF-stimulated MTLn3 carcinoma cells, Ras is required for both PtdIns(3,4,5)P(3) production and lamellipod extension, whereas Rac1 is required for formation of adhesive structures. These data suggest an unappreciated role for Ras during protrusion, and a crucial role for Rac in the stabilization of protrusions required for cell motility.

| S-EPMC4267689 | biostudies-literature

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